乳腺癌耐药细胞ER表达状态影响细胞对屈洛昔芬和阿霉素的敏感性

背景与目的：雌激素受体（estrogen receptor,ER)阳性乳腺癌细胞株来源的耐药细胞株中,ER表达缺失或下降,且细胞生长速度减慢,本文的目的是研究MCF－7／Adr乳腺癌耐药细胞株中ER表达状态与细胞对出洛昔芬（droloxifene,Dro)和阿霉素（Adriamycin,Adr)敏感性之间的关系。方法：Western blot法检测MCF－7／Adr及其亲本MCF－7细胞中ER蛋白的表达,构建ER的真核细胞表达质粒（pCER),利用LipofectAMINE^TM将ER基因导入MCF－7／Adr细胞,经G418抗性筛选获得阳性克隆（MTER／Adr),PCR,Western blot法鉴定并检测ER基因的整合和蛋白表达,流式细胞仪检测细胞周期变化,MTT法检测Dro及Adr对细胞增殖的影响。结果：在MCF－7细胞中可检测到ER蛋白 的表达,而MCF－7／Adr细胞中使用Westernblot检测不到ER蛋白的表达,成功构建真核细胞表达质粒pCER并转染MCF－7／Adr细胞,阳性克隆MTER／Adr整合了ER基因并获得表达。经MTT分析,10μmol/LDro对MCF－7细胞的生长有明显的抑制作用。而到20μmol/L时对MCF－7／Adr细胞的生长有抑制作用。ER转染MCF－7／Adr细胞后,15μmol/L的Dro对其生长出现抑制作用,使细胞多分布于G0／G1期。同时细胞对Adro的敏感性下降,结论：MCF－7／MCF－7／Adr细胞部分恢复对Dro和Adr的敏感性。

Status of estrogen receptor affects the drug sensitivity of drug-resistant MCF-7/Adr human breast cancer cells to droloxifene and Adriamycin

BACKGROUND & OBJECTIVE: Loss or decreased expression of estrogen receptor (ER) and decreased growth rate regularly occur in drug-resistant breast cancer cells. This study was designed to investigate the effect of estrogen receptor status on the drug resistance to droloxifene (Dro) and Adriamycin (Adr) of drug-resistant MCF-7/Adr human breast cancer cells. METHODS: The expression of ER in MCF-7 and MCF-7/Adr cells was determined using Western blot analysis. ER expression plasmid was constructed and introduced into MCF-7/Adr cells using LipofectAMINE. After G418 screening, the positive clone (MTER/Adr) was obtained. The integration and expression of ER gene were analyzed by polymerase chain reaction (PCR) and Western blot. The cell cycle distribution was investigated by flow cytometry. The effects of droloxifene and Adriamycin on the growth of cells were investigated by MTT assay. RESULTS: Western blot analysis showed that ER was positive in MCF-7 cells, but was negative in MCF-7/Adr cells. The ER expression plasmid was constructed and introduced into MCF-7/Adr cells. The integration and expression of ER gene were successful in positive clone -MTER/Adr cells. Droloxifene inhibited the growth of MCF-7 at the concentration of 10-20 micromol/L and the MCF-7/Adr only at concentration of 20 micromol/L. Droloxifene inhibited the growth of MTER/Adr at the concentration of 15 micromol/L, and the percentage of MTER/Adr cells increased in G0/G1 phase. The sensitivity of MTER/Adr cells to Adriamycin increased. CONCLUSION: The insensitivity of MCF-7/Adr human breast cancer cells to droloxifene was associated with the loss of ER. MTER/Adr cells partially restore the sensitivity to droloxifene and Adriamycin.